The molecular mechanisms involved in DNA replication and its regulation are being studied biochemically. To study properties of origin specific initiation of replication in vitro, I have developed an enzyme system that replicates exogenously added plasmid DNA containing the initiation origin of bacteriophage P1. The system requires the addition of the P1 replication protein, RepA, a partially purified protein fraction to supply E. coli replication proteins, ATP and an ATP regenerating system, dNTPs, rNTPs, Mg+2, buffer and polyvinyl alcohol. The specific E. coli replication proteins required include RNA polymerase, dnaA, dnaB, dnaC, dnaG, DNA pol III and DNA elongation accessory proteins and DNA gyrase. I have been studying the interaction of RepA and dnaA proteins with the P1 origin DNA. I have also been characterizing by deletion analysis the minimal DNA site required for initiation of replication. Since P1 normally exists stably as a unit copy plasmid, this system is being used to study the molecular mechanisms involved in the regulation of a stringently controlled replicon. A plasmid control locus, incA, is required for this regulated replication in addition to the origin region. It has been proposed that RepA is rate limiting for replication and that the role of incA is to sequester the initiator and thus reduce replication. This proposal appears inconsistent with the observation that RepA is autoregulated, since protein lost by sequestration should be replenished. A resolution of this autoregulation-sequestration paradox is possible if the sequestered RepA, unavailable for replication, is still available for promoter repression. In collaboration with D. Chattoraj (LB, NCI, NIH), we have shown that the RepA protein binds to incA and simultaneously to the repA promoter in the origin region, causing the intervening DNA to loop. DNA looping very likely could provide the mechanism by which RepA bound to incA might exert repression.